DTAT fusion toxin

ABSTRACT

The invention provides fusion toxins that contain one or more regions of diphtheria toxin and a portion of urokinase-type plasminogen activator, as well as the nucleic acids that encode the fusion toxins and methods of using the fusion toxins.

TECHNICAL FIELD

This invention relates to fusion toxins that are useful for targetingpathogenic cells. More particularly, this invention relates to fusiontoxins that are polypeptides containing a toxin domain, aninternalization domain, and a targeting domain that is a fragment of theurokinase-type plasminogen activator.

BACKGROUND

Toxin proteins such as diphtheria toxin (DT) typically are made up ofseveral functional domains, which may include a toxin (i.e., killing)domain, an internalization domain (e.g., the DT translocation enhancingregion (TER)), and a targeting domain to control recognition of andbinding to target cells. DT is a single chain of 535 amino acids(Greenfield et al. (1983) Proc. Natl. Acad. Sci. USA 80:6853-6857),which upon mild trypsinization and reduction in vitro breaks into an Achain and a B chain (Collier et al. (1971)J. Biol. Chem. 246:1496-503;and Moskaug et al. (1989)J. Biol. Chem. 264:15709-15713). The B chaincontains the targeting domain and the TER, which facilitatestranslocation of the A chain into the cytoplasm. Once in the cytoplasm,the toxin domain within the A chain catalyzes ADP-ribosylation of atranslationally modified histidine residue (diphthamide) on elongationfactor-2, leading to the arrest of protein synthesis and subsequent celldeath (Collier et al., ADP Ribosylation Reactions: Biology and Medicine,Academic Press, Inc., New York, p. 573 (1982)). Other toxins (e.g.,ricin) have similar A and B chain structures, while toxins such as thePseudomonas exotoxin have similar domains but in a single chainstructure.

Fusion toxins can be therapeutically useful in pathological conditionssuch as cancer, and particularly in types of cancer (e.g., certain braincancers) that are unresponsive to treatment by chemotherapy andradiation. Fusion toxins are chimeric polypeptides that typicallycontain a toxin protein or a toxin domain from a toxin protein, and atargeting domain from a heterologous protein (Kreitman (1999) Curr.Opin. Immunol. 11:570-578; and Oldfield and Youle (1998) Curr. Top.Microbiol. Immunol. 234:97-114). Fusion toxins may incorporate a portionof a toxin protein or an entire toxin protein (see, for example, Pastanand FitzGerald (1989) J. Biol. Chem. 264:15157-15160). Fusion toxinsthat contain an entire toxin molecule, however, typically result innon-specific killing mediated by binding to non-target cells. Fusiontoxins that contain only the toxin (killing) domain of a toxin protein,while much more specific, are much less toxic because they lack thetranslocation enhancing region that facilitates entry of the toxindomain into target cells.

Malignancies of the central nervous system are the third leading causeof cancer-related deaths among adolescents and adults from 15 to 34years of age (Davis et al. (1998) J. Neurosurg. 88:1-10). Patients withsuch malignancies typically have a two-year survival rate of less than20% (Thompson (1995) Science 267:1414). Although the anatomy of braintumors would especially lend them to intratumoral therapy with agentssuch as fusion toxins, therapeutic approaches to treating such tumorsare complicated by the fact that there has been no known tumor-specificmarker that can be targeted in the majority of patients (McKeever(1998)J. Histochem. Cytochem. 46:585-594).

SUMMARY

The present invention is based on the discovery that urokinase-typeplasminogen activator receptors (uPAR) are selectively overexpressed inglioblastoma multiforme, an aggressive form of brain cancer (Mori et al.(2000) J. Neuroonc. 46:115-123). In addition, uPAR expression iscorrelated with the invasive activity of glioma cells. uPAR also isoverexpressed in a number of other tumors, including cancers of thebreast, skin, colon, ovaries, thyroid, stomach, liver, and prostate (seeFabbrini et al. (1997) FASEB J. 11:1169-1176; and Rajagopal and Kreitman(2000) J. Biol. Chem. 275:7566-7573). Furthermore, uPAR is expressed onthe endothelial cells that make up tumor microvasculature.

The discovery that glioblastoma tumors overexpress uPAR allows fortargeting glioblastoma tumor cells with fusion toxins containing uPA,the ligand for these receptors. As described above, such fusion toxinswill be most useful if they incorporate the killing and translocationdomains of a toxin such as DT, while omitting the targeting domain ofthe toxin protein.

In one aspect, the invention provides a method for killing a tumor cell.The method includes contacting a tumor cell with a fusion toxincontaining the toxin domain of diphtheria toxin and a urokinase-typeplasminogen activator domain. The tumor cell can be a brain tumor cell(e.g., a glioblastoma, meningioma, astrocytoma, medulloblastoma,ependymoma, or oligodendroglioma cell). The tumor cell can express theurokinase-type plasminogen activator receptor (e.g., on its surface).The contacting of the tumor cell by the fusion toxin can occur in vivo.

The fusion toxin used to kill the tumor cell can contain thetranslocation enhancer region of diphtheria toxin. In anotherembodiment, the fusion toxin can contain the amino terminal 390 aminoacids of diphtheria toxin. The urokinase-type plasminogen activatordomain of the fusion toxin is capable of binding to urokinase-typeplasminogen activator receptor. The urokinase-type plasminogen activatordomain also can contain the amino terminal fragment of urokinase-typeplasminogen activator. In yet another embodiment, the fusion toxin cancontain the toxin domain of diphtheria toxin, the translocationenhancing region of diphtheria toxin, and the amino-terminal fragment ofurokinase-type plasminogen activator.

In another aspect, the invention provides a method for killing aglioblastoma tumor cell. The method includes contacting a glioblastomatumor cell with a fusion toxin containing a urokinase-type plasminogenactivator domain. The fusion toxin can contain a toxin domain of a toxinselected from the group consisting of diphtheria toxin, ricin,Pseudomonas exotoxin, colicin, anthrax toxin, tetanus toxin, botulinumneurotoxin, saporin, abrin, bryodin, pokeweed anti-viral protein,viscumin, and gelonin. In another embodiment, the fusion toxin also cancontain an internalization domain of a toxin selected from the groupconsisting of diphtheria toxin, colicin, delta-Endotoxin, anthrax toxin,tetanus toxin, botulinum toxin, and Pseudomonas exotoxin.

The urokinase-type plasminogen activator domain within the fusion toxinis capable of binding to urokinase-type plasminogen activator receptor.The urokinase-type plasminogen activator domain can contain theamino-terminal fragment of urokinase-type plasminogen activator. Theglioblastoma tumor cell that is contacted by the fusion toxin canexpress the urokinase-type plasminogen activator receptor (e.g., on itssurface). The fusion toxin can contain the toxin domain of diphtheriatoxin, the translocation enhancing region of diphtheria toxin, and theamino-terminal fragment of the urokinase-type plasminogen activator.

In another aspect, the invention provides a fusion toxin containing thetoxin domain of diphtheria toxin and a urokinase-type plasminogenactivator domain. The fusion toxin further can include the translocationenhancing region of diphtheria toxin. The urokinase-type plasminogenactivator domain of the fusion toxin can contain the amino-terminalfragment of urokinase-type plasminogen activator. In another embodiment,the fusion toxin can include the toxin domain of diphtheria toxin, thetranslocation enhancing region of diphtheria toxin, and theamino-terminal fragment of urokinase-type plasminogen activator.

In another aspect, the invention provides a pharmaceutical compositionthat contains the fusion toxin of the invention. The invention alsoprovides an article of manufacture that incorporates the pharmaceuticalcomposition of the invention.

In yet another aspect, the invention provides a nucleic acid containinga sequence that encodes the fusion toxin of the invention, as well asvectors containing the nucleic acid. The invention also provides hostcells containing such vectors and expressing a fusion toxin that cancontain the toxin domain of diphtheria toxin and a urokinase-typeplasminogen activator domain.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialsimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable materials andmethods are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

The details of one or more embodiments of the invention-are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a drawing of the pDTAT.pET21d expression plasmid.

FIGS. 2A and 2B are bar graphs showing the effect of DTAT on theviability of U118 MG glioblastoma cells after 48 hours of treatment(FIG. 2A) and 72 hours of treatment (FIG. 2B), versus treatment with aDThIL13 control.

FIG. 3 is a line graph depicting the effect of DTAT on the viability ofU87 MG glioblastoma cells, Neuro-2a neuroblastoma cells, SKBR3adenocarcinoma cells, and Daudi lymphoima cells.

FIG. 4 is a line graph showing the effect of DTAT on the viability ofU118 MG cells, versus treatment with a DTMIL4 control.

FIG. 5 is a line graph showing the response of U118 MG glioblastomacells to increasing doses of DTAT over 24, 48, and 72 hours oftreatment.

FIG. 6 is a line graph depicting the response of HUVEC to treatment withDTAT, as compared to DTmIL4, DThIL2, DTmIL13, and DThIL13 controls.

FIG. 7 is a bar graph showing the effect of anti-DTAT antibodies on theability of DTAT to kill HUVEC.

FIG. 8 is a line graph depicting tumor volumes in individual mice,before and after administration of DTAT.

FIG. 9 is a line graph depicting the average tumor size for groups ofmice treated with DTAT, PBS, DThIL2, or DThIL13.

FIGS. 10A and 10B are bar graphs showing serum levels of Blood UreaNitrogen (FIG. 10A) and alanine transferase (FIG. 10B) in mice injectedwith DTAT, PBS, or DTantiCD3sFv.

DETAILED DESCRIPTION

The present invention features fusion toxins that include at least onetoxin domain and at least one ligand for a cell surface receptor. Thefusion toxins may contain (a) a toxin domain (e.g., a portion of DT),(b) an internalization domain (e.g., the DT TER), and (c) a targetingdomain derived from the urokinase-type plasminogen activator (uPA). uPAis a ligand for the urokinase-type plasminogen activator receptor (UPAR)that is expressed, typically overexpressed, on the surfaces of certaintumor cells. As used herein, “overexpressed” means expressed at higherlevels than normally would be observed. For example, the levels of uPARexpressed on glioblastoma cells typically are higher than the levels ofuPAR expressed on corresponding non-tumorigenic brain cells. In anotherexample, the amount of DTAT produced by a cell transformed with a DTATexpression vector typically is greater than the amount produced by anuntransformed cell.

In one embodiment, fusion toxins according to the present invention maycontain the DT toxin domain, the DT internalization domain (TER), andthe amino-terminal fragment (ATF) of uPA. This embodiment of theinvention, termed DTAT, is particularly useful. Fusion toxins of thepresent invention can specifically inhibit the proliferation ofglioblastoma cell lines in vitro; the fusion toxins also can inhibit thegrowth of endothelial cells in vitro. Additionally, fusion toxins of theinvention can cause regression of human glioblastoma tumors in nudemice. Furthermore, the fusion toxins do not severely affect renal orhepatic function. uPAR therefore can serve as a valid target for therapyagainst glioblastoma tumors that are resistant to chemotherapy andradiation, and against the endothelial vasculature that feeds suchtumors.

Fusion Toxins

As used herein, “polypeptide” refers to an amino acid chain, regardlessof length or post-translational modification. A “functional fragment” ofa polypeptide refers to a fragment of the polypeptide that is shorterthan the full-length, wild-type polypeptide, but: which has at least 10%(e.g., 10%, 25%, 50%, 70%, 85%, 100%, or more) of the activity of thefull-length, wild-type polypeptide.

The fusion toxins of the present invention are polypeptides that containthe toxin domain of a toxin protein and a heterologous targeting domainderived from uPA. An internalization domain may also be included. Asused herein, “toxin domain” refers to a polypeptide or functionalfragment thereof that mediates a cytotoxic effect on a cell. Suitabletoxin domains can be, by way of example and not limitation, the toxindomains of DT, ricin, Pseudomonas exotoxin, colicin, anthrax toxin,tetanus toxin, botulinum neurotoxin, saporin, abrin, bryodin, pokeweedanti-viral protein, viscumin, and gelonin. The DT toxin domain isparticularly useful. Functional fragments of the DT A chain that containthe toxic function of DT also are particularly useful. Furthermore,fusion toxins of the invention may include more than one (e.g., 2, 3, 4,or more) toxin domains or functional fragments thereof. When multipletoxin domains are included, they may be immediately adjacent to eachother, separated by one or more targeting domains or internalizationdomains, or separated by a linker or linkers.

The amino acid sequence of a toxin domain of the invention can beidentical to the wild-type sequence of the naturally occurring toxindomain (e.g., the DT toxin domain). Alternatively, a toxin domain cancontain amino acid deletions, additions, or substitutions, provided thatthe toxin domain has at least 10% (e.g., 10%, 25%, 50%, 70%, 85%, 100%,or more) of the ability of the wild-type polypeptide to kill relevanttarget cells. In vitro and in vivo methods for comparing the relativeactivities of two or more toxins, whether naturally occurring toxins orfusion toxins, are known to those skilled in the art. Amino acidsubstitutions typically will be conservative substitutions, althoughnon-conservative substitutions (e.g., deletions and insertions) also arepossible. Conservative substitutions generally have little effect on thehydrophobicity of the polypeptide or the bulk of residue side chains,and typically include substitutions within the following groups: glycineand alanine; valine, isoleucine, and leucine; aspartic acid and glutamicacid; asparagine, glutamine, serine and threonine; lysine, histidine andarginine; and phenylalanine and tyrosine.

The fusion toxins of the present invention may contain aninternalization domain. As used herein, “internalization domain” refersto a polypeptide or functional fragment thereof that confers the abilityto translocate through the cell surface, across the cellular membraneand into the cytoplasm. An internalization domain typically is criticalfor the function of a fusion toxin, since a toxin cannot kill a cellunless it is internalized. Suitable internalization domains include anypolypeptide that mediates transfer of a protein through the cellularmembrane.

The internalization domain of DT (i.e., the TER), which is part of theDT B chain, is particularly useful for the fusion toxins of theinvention. Alternatively, fusion toxins may include internalizationdomains from other bacterial toxins that are adept at opening channelsin the lipid bilayer of the cellular membrane in order to facilitatetranslocation. These include, by way of example and not limitation,colicin, delta-Endotoxin, anthrax toxin, tetanus toxin, botulinumneurotoxin, and Pseudomonas exotoxin. Fusion toxins therefore canincorporate the internalization domain of any such molecule.

In one embodiment, the fusion toxins of the invention include both thetoxin domain and the internalization domain of DT. For example, as withDTAT, a fusion toxin may contain the N-terminal 390 amino acids of DT,which contain the toxin domain and the TER. Alternatively, a fusiontoxin may contain only those regions within the N-terminal 390 aminoacids that actually confer the killing and internalization functions.

Fusion toxins of the invention also include a targeting domain thatcontains uPA or a portion of uPA. As used herein, “targeting domain”refers to a polypeptide or functional fragment thereof that hassignificant binding affinity for a target molecule on the surface of atarget cell. In immunotoxins, for example, the target molecule is a cellsurface antigen and the targeting domain is an antibody. For fusiontoxins such as DTAT, the target is a cell surface receptor and thetargeting domain is a ligand for the receptor. According to the presentinvention, the targeting domain contains uPA or any portion of uPAcapable of binding to uPAR on the surfaces of target cells. The ATF ofuPA is particularly useful. The ATF includes the receptor-binding domainof uPA but does not include the catalytic or internalization domains.

Fusion toxin targeting domains of the invention may include the entireATF (i.e., the amino-terminal 135 amino acids of uPA.) These residuesinclude the receptor-binding domain, which is an epidermal growthfactor-like domain situated between amino acids 12 and 32, and whichbinds to uPAR with high affinity (K_(d)=9.5 nM). Alternatively, fusiontoxins may contain a targeting domain that is a functional fragment ofthe uPA ATF.

Fusion toxin targeting domains of the present invention can have aminoacid sequences that are identical to the wild-type sequence of the uPAATF. Alternatively, a targeting domain may contain amino acid deletions,additions, or substitutions, provided that the targeting domain has atleast 10% (e.g., 10%, 25%, 50%, 70%, 85%, 100%, or more) of the abilityof the wild-type uPA polypeptide to bind to the target molecule. Methodsof comparing the relative ability of two of more molecules (e.g., fusiontoxins or ligands) to bind to a target cell are well known in the art.Amino acid substitutions typically will be conservative substitutions(see above), although non-conservative substitutions are possible.

The toxin domains, internalization domains, and targeting domains withinthe fusion toxins of the invention can be positioned in any orientationwith respect to each other. For example, a toxin domain can beN-terminal of an internalization domain and a targeting domain, or atargeting domain or an internalization domain can be at the N-terminus.The three domains can be immediately adjacent to each other and coupledvia peptide bonds, or they can be coupled via a linker or linkers. Inone embodiment, such linkers can be peptides (in which case the couplingis again via peptide bonds, as in DTAT). Alternatively, cross-linkingagents or hetero bifunctional cross-linking agents (e.g., cystamine,m-Maleimidobenzoyl-N-hydroxysuccinimide-ester,N-succinimidyl-3-(2-pyridyldithio)-propionate,methylmercaptobutyrimidate, or dithiobis(2-nitrobenzoic acid) can beutilized to generate disulfide or thioether bonds between the domains.

The fusion toxins of the invention also can be modified for use in vivoby the addition, at the amino- or carboxy-terminal ends, of a blockingagent to facilitate survival of the fusion toxins in vivo. This can beuseful in situations in which peptide termini tend to be degraded byproteases prior to cellular uptake. Such blocking agents can include,without limitation, additional related or unrelated peptide sequencesthat can be attached to the amino- and/or carboxy-terminal residues ofthe fusion toxins to be administered. Blocking can be achieved eitherchemically during the synthesis of the fusion toxins or by recombinantDNA technology using methods familiar to those of ordinary skill in theart. Alternatively, blocking agents such as pyroglutamic acid or othermolecules known in the art can be attached to the amino- and/orcarboxy-terminal residues, or the amino group at the amino terminus orthe carboxyl group at the carboxy terminus can be replaced with adifferent moiety.

Fusion toxins of the present invention (e.g., DTAT) can be produced bystandard methods, combining in vitro recombinant DNA techniques toproduce vectors comprising nucleotide sequences that encode the fusiontoxins, overexpression of the fusion toxins in host cells, andbiochemical purification of the resulting cellular extracts. Suchexpression vectors, containing relevant coding sequences and appropriatetranscriptibnal/translational control signals, can be contstructedusing, for example, methods well known to those of ordinary skill in theart. See, for example, techniques described in Sambrook et al.,Molecular Cloning: A Laboratory Manual (2nd Ed.), Cold Spring HarborLaboratory, New York (1989); and Ausubel et al., Current Protocols inMolecular Biology, Green Publishing Associates and Wiley Interscience,New York (1989). See also the following subsection and Example 1, below.

Expression systems that can be used for small or large scale productionof the fusion toxins of the invention include, but are not limited to,microorganisms such as bacteria (e.g., E. coli and B. subtilis)transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmidDNA expression vectors containing the nucleic acid molecules of theinvention; yeast (e.g., S. cerevisiae) transformed with recombinantyeast expression vectors containing the nucleic acid molecules of theinvention; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing the nucleic acidmolecules of the invention; plant cell systems infected with recombinantvirus expression vectors (e.g., tobacco mosaic virus) or transformedwith recombinant plasmid expression vectors (e.g., Ti plasmid)containing the nucleic acid molecules of the invention; or mammaliancell systems (e.g., COS, CHO, HeLa, 293, and 3T3 L1 cells) harboringrecombinant expression constructs containing promoters derived from thegenome of mammalian cells (e.g., the metallothionein promoter) or frommammalian viruses (e.g., the adenovirus late promoter or thecytomegalovirus promoter), along with the nucleic acid molecules of theinvention. Also useful as host cells are primary or secondary cellsobtained directly from a mammal, transfected with a plasmid vector orinfected with a viral vector that contains the nucleic acids of theinvention.

Nucleic Acids

The invention provides nucleic acids encoding the above polypeptides ofthe invention. As used herein, the term “nucleic acid” refers to bothRNA and DNA, including cDNA, genomic DNA, and synthetic (e.g.,chemically synthesized) DNA. The nucleic acid can be double-stranded orsingle-stranded (i.e., a sense or an antisense single strand). Nucleicacids of the invention may have sequences identical to those of nucleicacids encoding the wild-type toxin, internalization, and targetingdomains that are incorporated into the fusion toxins of the invention.Alternatively, nucleic acids of the invention may contain codons otherthan wild-type codons which, due to the degeneracy of the genetic code,encode toxin, internalization, or targeting domains with amino acidsequences identical to relevant wild-type polypeptides. Furthermore, thenucleic acids may encode toxin, internalization, or targeting domainsthat are not identical to the wild type polypeptide due to the presenceof, for example, any of the above described deletions, additions, orsubstitutions.

Nucleic acids of the invention may be hybrid genes. As used herein,“hybrid gene” refers to a nucleic acid molecule that encodes amino acidsequences from more than one polypeptide. Hybrid genes of the inventiontypically will contain a first portion and a second portion, and maycontain more portions (e.g., a third portion, a fourth portion, or moreportions). For example, a first portion may encode the DT toxin domain,a second portion may encode an internalization domain, and a thirdportion may encode a targeting domain comprising the uPA ATF. Theseportions may be arranged in any order relative to one another, andbetween any of the portions can be codons encoding a linker (see aboveand Example 1).

The invention also provides vectors containing nucleic acids such asthose described above. As used herein, a “vector” is a replicon, such asa plasmid, phage, or cosmid, into which another DNA segment may beinserted so as to bring about the replication of the inserted segment.The vectors of the invention typically are expression vectors. An“expression vector” is a vector that includes expression controlsequences, and an “expression control sequence” is a DNA sequence thatcontrols and regulates the transcription and translation of another DNAsequence.

In the expression vectors of the invention, the nucleic acid sequenceencodes a fusion toxin with an initiator methionine, operably linked toone or more transcriptional regulatory elements. As used herein,“operably linked” means incorporated into a genetic construct so thatexpression control sequences effectively control expression of a codingsequence of interest. Examples of transcriptional regulatory elementsinclude promoters, enhancers, and transcription terminating regions. Apromoter is a transcriptional regulatory element composed of a region ofa DNA molecule, typically within 100 nucleotides upstream of the pointat which transcription starts (generally near the initiation site forRNA polymerase II). To bring a coding sequence under the control of apromoter, it is necessary to position the translation initiation site ofthe translational reading frame of the polypeptide between one and aboutfifty nucleotides downstream of the promoter. Enhancers provideexpression specificity in terms of time, location, and level. Unlikepromoters, enhancers can function when located at various distances fromthe transcription site. An enhancer also can be located downstream fromthe transcription initiation site. A coding sequence is “operablylinked” and “under the control” of transcriptional and translationalcontrol sequences in a cell when RNA polymerase is able to transcribethe coding sequence into mRNA, which then is translated into the proteinencoded by the coding sequence.

Suitable expression vectors include, without limitation, plasmids andviral vectors derived from, for example, bacteriophage, baculoviruses,tobacco mosaic virus, herpes viruses, cytomegalovirus, retroviruses,vaccinia viruses, adenoviruses, and adeno-associated. viruses. Numerousvectors and expression systems are commercially available from suchcorporations as Novagen (Madison, Wis.), Clontech,(Palo Alto, Calif.),Stratagene (La Jolla, Calif.), and Invitrogen/Life Technologies(Carlsbad, Calif.).

The expression vectors of the invention, which contain nucleic acidsequences encoding fusion toxins, have a variety of uses. For example,they can be used to transform or transfect either prokaryotic (e.g.,bacteria) or eukaryotic (e.g., yeast, plant, insect, or mammalian)cells. Such cells then may be used for small or large scale in vitroproduction of fusion toxins by methods such as those known in the art(see preceding subsection, above, and Example 1, below). These methodsmay involve, for example, culturing the cells under conditions thatmaximize production of the fusion toxin, and isolating the fusion toxinfrom the cells or from culture medium. Transformed/transfected cellsalso can be used for delivery of a fusion toxin to a target cell byadministration of the transformed/transfected cells to the target cells.

Methods for Using Fusion Toxins

The fusion toxins of the invention can be administered to a cellpopulation in order to kill those cells that have surface expression ofthe target molecule for the targeting domain. Fusion toxins. (e.g.,DTAT) can be administered as therapeutic agents, for example, when it isdesired to eliminate a cell population (e.g., a tumor) that expressesthe uPAR target. Furthermore, fusion toxins can be administered eitherex vivo or in vivo.

In one embodiment, fusion toxins of the present invention can beadministered to target cells derived from tumors. Through such an exvivo approach, the effectiveness of various preparations of the fusiontoxins can be evaluated before they are used for in vivo approaches.Methods for quantifying the toxicity of one or more fusion toxins areknown to those skilled in the art (e.g., as in Example 1).

In another embodiment, fusion toxins such as DTAT can be incorporatedinto pharmaceutical compositions and administered to a subject thatexhibits tumor growth. Tumors suitable for treatment by fusion toxinssuch are DTAT include, by way of example and not limitation,glioblastoma, meningioma, astrocytoma, medulloblastoma, ependymoma, andoligodendroglioma. Generally, pharmaceutical compositions of theinvention contain one or more fusion toxins suspended in apharmaceutically acceptable carrier. A “pharmaceutically acceptablecarrier” is a pharmaceutically acceptable solvent, suspending agent, orany other pharmacologically inert vehicle for delivering one or moretherapeutic compounds (e.g., fusion toxins such as DTAT) to a subject.Pharmaceutically acceptable carriers can be liquid or solid, and can beselected with the planned manner of administration in mind so as toprovide for the desired bulk, consistency, and other pertinent transportand chemical properties, when combined with one or more of therapeuticcompounds and any other components of a given pharmaceuticalcomposition. Typical pharmaceutically acceptable carriers include, byway of example and not limitation: water; saline solution; bindingagents (e.g., polyvinylpyrrolidone or hydroxypropyl methylcellulose);fillers (e.g., lactose and other sugars, gelatin, or calcium sulfate);lubricants (e.g., starch, polyethylene glycol, or sodium acetate);disintegrates (e.g., starch or sodium starch glycolate); and wettingagents (e.g., sodium lauryl sulfate).

Fusion toxins of the invention can be administered in any of a number ofways. For example, fusion toxins may be administered orally or byintravenous infusion, or they may be injected subcutaneously,intramuscularly, intratumorally, intraperitoneally, intrarectally,intravaginally, intranasally, intragastrically, intratracheally, orintrapulmonarily. Intratumoral injection is particularly useful, sinceit allows a fusion toxin to be delivered directly to the appropriatetissue (e.g., brain tissue where tumor growth is occurring). Such directdelivery can results in concentration of a fusion toxin at the affectedtissue, while avoiding or greatly reducing systemic effects onnon-target tissues. The dosage required will depend on the route ofadministration, the nature of the composition, the nature of thesubject's illness, and the subject's size, weight, surface area, age,and sex, other drugs being administered, and the judgment of theattending physician. Wide variations in the necessary dosage are to beexpected in view of the differing efficiencies of various routes ofadministration. For example, oral administration would be expected torequire higher dosages than administration by injection. Variations inthese dosage levels can be adjusted using standard empirical routinesfor optimization as is well understood in the art.

Articles of Manufacture

Fusion toxins of the invention can be combined with packaging materialand sold as kits for killing tumor cells. Components and methods forproducing articles of manufacture such as kits are well known. Anarticle of manufacture may include one or more of the fusion toxins setout in the above sections. In addition, the article of manufacturefurther may include buffers or other solutions necessary to effect tumorcell death. Instructions describing how the fusion toxins are effectivefor killing target cells can be included in such kits.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 Materials and Method

Plasmid construction: The pDTAT.pET21d expression plasmid (FIG. 1) wasconstructed by ligating a C-terminally truncated fragment of DT and theATF of uPA, and subcloning the ligation product into pET21d (Promega,Madison, Wis.). To clone the individual cDNA segments with appropriaterestriction sites, the cDNA sequence encoding human mature urokinase(Accession No: E01560) and the gene sequence encoding diphtheria toxin(Corynebacteriophage beta (C. diphtheriae) gene and flanks; AccessionNo: K01722) were obtained from GenBank. The uPA ATF was assembled bysynthesizing 20 oligonucleotides, each 40 bp in length, which coveredboth strands over the length of the ATF. Oligonucleotides were designedto place a HindIII restriction site at the 5′ end of the assembly,followed by sequences encoding a linker with the amino acid sequenceGluAlaSerSerGlyGlyProGlu (SEQ ID NO:1). Oligonucleotides containing astop codon followed by a XhoI site were positioned at the 3′ end of theassembly. The pooled oligonucleotides were used as templates for eachother in a single PCR reaction. The amplified product was purified froma 1% agarose gel and subcloned into the pGEM®-T Easy vector (Promega).The fidelity of the product was verified by DNA sequencing (AdvancedGenetic Analysis Center, University of Minnesota, St. Paul, Minn.).Mutations were corrected with the QuikChange™ Site-Directed MutagenesisKit (Stratagene, La Jolla, Calif.). A gene fragment encoding the first390 amino acids of diphtheria toxin (389 amino acids plus a methioninestart codon) was assembled in an identical fashion and then ligated intothe prokaryotic expression vector pET21d at the NcoI and HindIII sitesto generate DT₃₉₀.pET21d. The HindIIIIXhoI ATF uPA fragment subsequentlywas subcloned into the DT₃₉₀.pET21d construct to generate DTAT.pET21d(FIG. 1), which encodes the recombinant fusion toxin, DTAT. DTATtherefore consists of DT₃₉₀ and the 135 amino acid ATF of uPA. The DTATprotein product had a predicted MW of 58.1 kDa and an isoelectric pointof 6.073.

DTAT expression and purification: The pDTAT.pET21d expression plasmidwas transformed into the E. coli strain BL21 (DE3) (Novagen, Madison,Wis.). Recombinant bacteria were grown on LB plates containingcarbenicillin (Sigma, St. Louis, Mo.) at 37° C. for 18 hours. Colonieswere scraped and distributed into four 1 L superbroth culturessupplemented with 100 μg/ml carbenicillin, and grown in a 2 L flask at37° C. When the absorbance (A₆₀₀) reached 0.8, expression of the hybridgene was induced by the addition of 1 mMisopropyl-β-D-thiogalactopyranoside (IPTG, Gibco/BRL). Twenty-one and ahalf hours after induction, cells were harvested by centrifugation at32,000 g for 10 minutes. All four bacterial pellets were resuspended inTE/NaCl (50 mM Tris pH 8.0, 20 mM EDTA, 100 mM NaCl), pooled in a totalvolume of 200 ml, and transferred into a rosette sonication cup (Sonicsand Material Inc., Newton, Conn.). The bacteria were lysed by sonicationat 4° C. for 30 minutes. Inclusion bodies were spun down at 14,000 g for10 minutes and were extracted for 18 hours at 4° C. with 150 ml TE/NaClcontaining 5% Triton X-100, 10% glycerin, and 0.3% sodium deoxycholate.The pellet was washed twice with detergent, followed by three washeswith TE/NaCl. Partially purified inclusion bodies were solubilized to 10mg/ml with 0.1 M Tris pH 8.0, 7 M guanidine-HCl, 64 mM DTT, and 2 mMEDTA. Renaturation was initiated by 100-fold dilution of the denaturedprotein into chilled refolding buffer containing 0.1 M Tris pH 8.0, 0.5M L-arginine, 0.9 mM GSSG (Calbiochem, San Diego, Calif.) and 2 M EDTA.

Samples were incubated at 4° C. for 48 hours. The refolded protein wasfiltered through a 0.45μ filter and then diluted 10-fold with distilledwater. This refolded and diluted protein was purified further passingthe samples over a Fast Flow Q-Sepharose column (Sigma) and eluting theprotein with a 0 to 1 M NaCl gradient in 20 mM Tris pH 8.0. The DTATfusion toxin eluted at 0.2 to 0.3 M NaCl, and residual contamination wasremoved by size-exclusion chromatography on a TSK 250 column (TosoHass,Philadelphia, Pa.), using SDS-PAGE to assess each fraction. The activepeak was pooled, concentrated, and dialyzed against PBS.

Cell lines and antibodies: The U118 MG, U87 MG, U373 MG, and T98 G celllines were derived from human patients diagnosed with glioblastomamultiforme and were provided by Dr. Walter Low, University of Minnesota.The Neuro-2a (murine neuroblastoma), Daudi (human Burkitt's lymphoma),and SKBR3 (human mammary gland adenocarcinoma) lines were obtained fromAmerican Type Culture Collection (Manassas, Va.). All cell lines weremaintained in RPMI 1640 medium supplemented with 10% heat-inactivatedfetal bovine serum (both from Biowhittaker, Walkersville, Md.), and 2 mML-glutamine, 0.1 mM non-essential amino acids, 1.0 mM sodium pyruvate,and 100 U/ml penicillin/100 μg/ml streptomycin (all from Gibtco/BRL).Human umbilical vein endothelial cells (HUVEC) were obtained from Dr. S.Ramakrishnan, University of Minnesota. HUVEC were maintained in Medium199 (Gibco/BRL) containing 15% heat-inactivated fetal bovine serum, 100U/ml penicillin/100 μg/ml streptomycin, and Endothelial Cell GrowthMedium (Biowhittaker), and were used within seven passages. All cellswere maintained at 37° C. in a humidified incubator in 5% CO₂/95% air,and passaged two to three times per week.

Polyclonal rabbit anti-human urokinase IgG and a murine IgG2a monoclonalantibody against human urokinase receptor were obtained from AmericanDiagnostica Inc. (Greenwich, Conn.), and were used for blockingexperiments. Anti-IL-4 (rat anti-mouse IgG1) was obtained from clone11B11 (American Type Culture Collection, Manassas, Va.).

Cytotoxicity assay: The in vitro cytotoxicity of DTAT was tested bymeasuring the incorporation of radiolabeled thymidine into DNA intreated and untreated cells, using a previously described method(Vallera et al. (1997) Protein Eng. 10:1071-1076). 10⁴ cells in 100 μlmedium were seeded per well on 96-well plates. Following an overnightincubation, cells were cultured in the presence of variousconcentrations of DTAT for 24, 48, or 72 hours. 1 μCi[methyl-³H]-thymidine (Amersham Pharmacia Biotech UK Limited,Buckinghamshire, England) was added at the beginning of the final 8hours of incubation. At the end of the incubation, cells were washed andharvested on glass fiber filters, and the incorporation of radioactivitywas quantified. Assays were performed in triplicate and repeated 2 to 4times; data is expressed as the percentage of [³H]-thymidineincorporation in cells incubated without toxin. Statistical significancewas assessed by Student's t test. For blocking experiments, cells ortoxins were preincubated with antibodies for 30 minutes at 37° C. andthe assay was then performed as above.

In vivo studies: Female athymic nu/nu nude mice and C57BL/6 mice 6 to 8weeks of age were purchased from NIH (Bethesda, Md.), and weremaintained in microisolator cages under specific pathogen-freeconditions by the Department of Research Animal Resources, University ofMinnesota, Minneapolis, Minn. On days -2 and -4, 25 μl anti-asialoGMI(Wako Chemicals USA, Richmond, Va.) diluted in 175 μl PBS was injectedintraperitoneally into each mouse to enhance tumor growth. On day 0,6×10⁶ U118 MG cells in 0.1 ml culture medium were injectedsubcutaneously into the right flank of each mouse. Each treatment groupconsisted of 4 to 5 animals and mice were examined every 2 to 3 days.Palpable tumors larger than 0.15 cm³ were treated by intratumoralinjection beginning on day 28, such that each mouse received aninjection of 20 μg DTAT in 50 μl PBS every other day over the course of9 days (for a total of 5 injections). Control animals receivedinjections of DThIL2 or PBS. Tumor volumes were monitored by calipermeasurement of length, width, and height.

Human glioblastoma tumors also were established in the brains of nudemice. Animals were restrained in a rat stereotactic head frame (DavidKopf Instruments, Tujunga, Calif.) and intracranially injected with1×10⁵ U87 MG cells in 3 μl culture medium on study day 0. Initial MRIscans were performed on day 15 post tumor injection, at which time micewere treated with either 5 μg DTAT in 3 μl PBS or PBS alone, injected tothe same stereotactic coordinates as the tumor cells. The effects oftreatment with DTAT or PBS were determined by MRI scans on day 22 posttumor injection. MRI scans were performed using a Philips 1.5 Tesla MRIscanner (Philips Medical Instruments, Bothell, Wash.).

Histology: Mice were sacrificed and tissues were removed forhistopathologic analysis as previously described (Vallera et al.,above). Samples were imbedded in OCT (Miles, Elkhart, Ind.), snap frozenin liquid nitrogen, and stored at −80° C. until sectioning. Serial 4 μmsections were thaw mounted onto glass slides, fixed for 5 minutes inacetone, and stained with hematoxylin and eosin for assessment.

Blood Urea Nitrogen (BUN) and alanine transferase (ALT) assays: Bothassays were performed as previously described (Vallera et al. (2000)Cancer Res. 60:976-984) on a Kodak ETACHEM 950 by the FairviewUniversity Medical Center—University Campus (Minneapolis, Minn.). Micewere sacrificed, serum samples were collected by heart bleeding, andanalyses were performed blindly on undiluted samples. The minimum samplevolume for each assay was 11 μl. BUN was measured spectrophotometricallyat 670 nm, while the oxidation of NADH was used to measure ALT activityat 340 nm.

Example 2 Effectiveness of DTAT In Vitro

To determine whether an IL13/diphtheria fusion protein would kill humanglioblastoma cells, DThIL13 was used in cytotoxicity assays as describedin Example 1. DThIL13 completely blocked DNA synthesis in the U373 MGline, with an IC₅₀ less than 0.01 nM. In contrast, DNA synthesis in theU87 MG line was inhibited only 45% by 10 nM DThIL13, and the T98 G linewas completely unaffected, demonstrating that DThIL13 does not kill allglioblastoma cells. Since uPAR is overexpressed in certain cancers, theeffectiveness of DTAT was tested using the U118 MG glioblastoma line.DNA synthesis in these cells was inhibited by approximately 94% after 48hours of treatment with 10 nM DTAT, while DThIL13 resulted in only a 73%reduction (FIG. 2A). At 72 hours, DTAT had blocked more than 94% of DNAsynthesis in the U118 MG cells (FIG. 2B). The difference between DTATand DTHIL13 treatment was statistically significant at both time points(P<0.05).

To study the selectivity of DTAT, it was tested in various cell linesthat do not express uPAR. DNA synthesis in the U87 MG glioblastoma linewas completely inhibited by treatment with 10 nM DTAT for 72 hours(IC₅₀<1 nM), while the Daudi lymphoma and SKBR3 mammary glandadenocarcinoma lines were not affected (FIG. 3). DNA synthesis in theNeuro2a murine neuroblastoma line was partially inhibited. To furtherstudy selectivity, U118 MG cells were treated with a mouseIL4/diphtheria fusion toxin, used as a negative control since mouse IL4is species specific and does not bind to human cells. Whereas 10 nM DTATinhibited DNA synthesis in U118 MG cells after 72 hours, DTmIL4 had noeffect (FIG. 4). DTAT is therefore highly selective in its ability tokill receptor-expressing cells.

To determine whether exposure of DTAT to its target for longer periodswould increase its effectiveness, various concentrations of the fusiontoxin were incubated with U118 MG cells for 24, 48, and 72 hours. Thesedose response/time course studies revealed that maximal toxicity wasobserved after 48 hours, and exposure to DTAT for 72 hours did notenhance cytotoxicity (FIG. 5).

As mentioned above, the ability of DTAT to bind to a tumor and itsmicrovasculature could provide a therapeutic advantage since tumorgrowth is dependent on a thriving vasculature. To assess theeffectiveness of DTAT on vascular cells, the fusion toxin was incubatedwith HUVEC. DTAT was able to inhibit the proliferation of HUVEC in adose dependent manner, with an IC₅₀ of about 1 nM after 72 hours oftreatment (FIG. 6). In contrast, a number of control fusion toxins thatbind to receptors not found on HUVEC (including mIL4R, hIL2R, hIL13R,and mIL13R) were much less cytotoxic.

To ascertain whether the activity of DTAT was mediated by the ATF, DTATwas preincubated with a monoclonal or polyclonal anti-urokinase antibodybefore administration to HUVEC, as described in Example 1. Suchtreatment completely blocked the cytotoxic effect of 1 nM DTAT andreduced the effectiveness of 10 nM DTAT, whereas a control anti-mouseIL4 antibody had no effect (FIG. 7). Together, these results demonstratethat DTAT selectively kills endothelial cells in vitro through itsability to bind uPAR.

Example 3 Effectiveness of DTAT In Vivo

To determine the effectiveness of DTAT in vivo, a nude mouse model ofhuman glioblastoma was established. U118 MG cells were inoculated intothe flanks of nude mice as described in Example 1. After 28 days, micewith established tumors were given a course of DTAT. In a test group offive mice, all tumors regressed following a 5 dose course of 20 μg DTATinjected directly into each tumor every other day. Tumors continued toshrink during the days following treatment such that by study day 62 allbut one tumor had completely regressed (FIG. 8). In contrast, tumors ingroups of mice treated with DThIL2, DThIL13, or PBS did not regress overthe study period, and in fact continued to increase in size (FIG. 9).There was a significant difference in the tumor growth curves on day 48when the DTAT group was compared to the control groups (P<0.05). Thesein vivo observations are correlated with the results of the in vitrotoxicity studies, and support the hypothesis that DTAT might be a usefultreatment for tumors not responsive to DTIL13.

To examine the effectiveness of DTAT for treating brain tumors, a secondmodel of human glioblastoma was established in nude mice. U87 MG cellswere injected intracranially as described in Example 1. MRI scans wereperformed on day 15 post injection to verify the presence of tumors,which were visualized in coronal MRI frames taken in 1 mm slices fromeach mouse. Two mice,then were treated with 5 μg DTAT, administered tothe same stereotactic coordinates as the tumor cells, while the thirdmouse was treated with PBS. MRI scans were repeated on day 22 post tumorinjection. The tumor in the control mouse continued to grow after PBStreatment. The mice treated with DTAT; however, displayed significanttumor regression. The tumor size was reduced approximately 95% in oneDTAT-treated mouse, while no evidence of tumor was found in the otherDTAT-treated mouse.

To assess potentially toxic effects of DTAT on areas outside tumors,control animals without tumors were subcutaneously injected with 20 μgDTAT every other day for a total of 5 doses and tissues sections andblood samples were obtained the day after the final injection. Frozensections were stained and serum analyses were performed as described inExample 1. Kidney tissue appeared unaffected by DTAT treatment, with theexception of some minor neutrophil infiltration. In addition, there wasno significant fluctuation in BUN levels following DTAT treatment (FIG.10A), indicating that DTAT does not interfere with renal activity. Thesestudies were particularly important because similar doses ofimmunotoxins in other studies had induced glomerular destruction,rupture of renal tubules, and proximal tubular vacuolization (Vallera etal. (1997), above). As a control, a separate group of mice was treatedwith DTanti-CD3sFv, a fusion toxin previously shown to mediate renaldamage. DTantiCD3sFv caused a significant elevation in BUN activity(FIG. 10A), and histology studies confirmed glomerular damage andtubular rhexis.

Histologic examination revealed that liver tissue was relativelyunaffected by DTAT treatment, other than in a few areas that exhibitedsmall amounts of infiltration. DTAT also had no effects on heart orspleen. Functional analysis revealed a significant, albeitnon-life-threatening elevation in ALT levels (FIG. 10B). Although thiselevation was not indicative of liver failure, and histology indicatedthat the liver was intact, it appeared that DTAT did affect the liver atthis dose. However, these studies were done in normal, non-tumorigenicmice. It is possible that a tumor and its extensive vascular networkcould act as an “antigenic sink” to absorb injected DTAT and limit theamount that would leave the tumor. Furthermore, the absence of this“antigenic sink” in the tumor-free control mice injected with DTAT mayhave resulted in greater toxic stress on the liver, thus explaining theelevated ALT levels. Preliminary experiments indicating that mice withlarge (>6 cm³) subcutaneous tumors were able to tolerate a 100 μg/dosecourse of DTAT provide support for this possibility.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A method for killing a tumor cell, comprising contacting said tumorcell with a fusion toxin comprising the toxin domain of diphtheria toxinand a urokinase-type plasminogen activator domain.
 2. The method ofclaim 1, wherein said tumor cell is a brain tumor cell.
 3. The method ofclaim 2, wherein said brain tumor is selected from the group consistingof glioblastoma, meningioma, astrocytoma, medulloblastoma, ependymoma,and oligodendroglioma.
 4. The method of claim 2, wherein said braintumor is a glioblastoma.
 5. The method of claim 1, wherein said tumorcell expresses the urokinase-type plasminogen activator receptor.
 6. Themethod of claim 1, wherein contacting said tumor cell occurs in vivo. 7.The method of claim 1, wherein said fusion toxin comprises thetranslocation enhancer region of diphtheria toxin.
 8. The method ofclaim 1, wherein said fusion toxin comprises the amino terminal 390amino acids of diphtheria toxin.
 9. The method of claim 1, wherein saidurokinase-type plasminogen activator domain is capable of binding tourokinase-type plasminogen activator receptor.
 10. The method of claim9, wherein said urokinase-type plasminogen activator domain comprisesthe amino terminal fragment of urokinase-type plasminogen activator. 11.The method of claim 1, wherein said fusion toxin comprises the toxindomain of diphtheria toxin, the translocation enhancing region ofdiphtheria toxin, and the amino-terminal fragment of urokinase-typeplasminogen activator.
 12. A method for killing a glioblastoma tumorcell, comprising contacting said glioblastoma tumor cell with a fusiontoxin comprising a urokinase-type plasminogen activator domain.
 13. Themethod of claim 12, wherein said fusion toxin comprises a toxin domainof a toxin selected from the group consisting of diphtheria toxin,ricin, Pseudomonas exotoxin, colicin, anthrax toxin, tetanus toxin,botulinum neurotoxin, saporin, abrin, bryodin, pokeweed anti-viralprotein, viscumin, and gelonin.
 14. The method of claim 12, wherein saidfusion toxin comprises the toxin domain of diphtheria toxin.
 15. Themethod of claim 12, wherein said fusion toxin comprises aninternalization domain of a toxin selected from the group consisting ofdiphtheria toxin, colicin, delta-Endotoxin, anthrax toxin, tetanustoxin, botulinum toxin, and Pseudomonas exotoxin.
 16. The method ofclaim 12, wherein said fusion toxin comprises the translocationenhancing region of diphtheria toxin.
 17. The method of claim 12,wherein said urokinase-type plasminogen activator domain is capable ofbinding to urokinase-type plasminogen activator receptor.
 18. The methodof claim 17, wherein said urokinase-type plasminogen activator domaincomprises the amino-terminal fragment of urokinase-type plasminogenactivator.
 19. The method of claim 12, wherein said glioblastoma tumorcell expresses the urokinase-type plasminogen activator receptor. 20.The method of claim 12, wherein said fusion toxin comprises the toxindomain of diphtheria toxin, the translocation enhancing region ofdiphtheria toxin, and the amino-terminal fragment of the urokinase-typeplasminogen activator. 21.-29. (canceled)